Biology has built up a vast set of highly complex and dynamic networks that govern nearly all cellular processes. Crucial to understanding these processes, fluorescent proteins (FPs) are the label of choice in characterizing protein location, interactions, and dynamics in live cells. The convenience afforded by FPs often outweighs challenges associated with auto fluorescent background, moderate brightness, and limited distinguishable colors. Our proposed studies will develop an important new class of emitters - optically modulated fluorescent proteins (OMFPs). By taking advantage of the unique timescale associated with each OMFP for the buildup of a fluorescence-limiting dark state population, we will be able to 1) separate desired signals from all (unmodulatable) background and 2) distinguish signals from multiple, otherwise spectrally indistinguishable emitters. Through targeted mutations that stabilize various chromophore states, we will tune the modulation depth vs. modulation frequency spectrum of each OMFP. The resulting unique modulation frequency responses will be utilized to expand the dimensionality of fluorescence imaging to simultaneously distinguish at least 3-fold more fluorophores within the same spectral regions, all while rejecting all unmodulatable auto fluorescent background. Multiple OMFPs will be produced in each color - blue through red, and within a given color, at least three spectrally similar emittr signals will be spatially resolved in live cells based on their unique modulation frequency responses. This spectral unmixing of modulation spectra, coupled with background removal through optical demodulation, will be generally applicable to simultaneously follow multiple emitters within a single spectral region. Benchmarking of sensitivities will be performed to demonstrate advantages of the designed materials and methods. We will employ these methods to simultaneously image up to 6 emitters in a single cell, and push methods to employ optical modulation for drastically improved sensitivity using commercial confocal microscopes. This effort will provide a general set of tools, methods, and overall framework for surveying and measuring multiple overlapping protein locations within the complex environment of live cells.